The major types of lipids that circulate in plasma include cholesterol and cholesteryl esters, phospholipids and triglycerides. Braunwald's Heart Disease, P. Libby, R. Bonow, D. Mann and D. Zipes, Eds., 8th Edition, Saunders Elsevier, Philadelphia, Pa. (2008) at 1071. Cholesterol contributes an essential component of mammalian cell membranes and furnishes substrate for steroid hormones and bile acids. Many cell functions depend critically on membrane cholesterol, and cells tightly regulate cholesterol content. Most of the cholesterol in plasma circulates in the form of cholesteryl esters in the core of lipoprotein particles. The enzyme lecithin cholesterol acyl transferase (LCAT) forms cholesteryl esters in the blood compartment by transferring a fatty acyl chain from phosphatidylcholine to cholesterol. Id.
Lipoproteins are complex macromolecular structures composed of an envelope of phospholipids and free cholesterol, a core of cholesteryl esters and triglycerides. Id. at 1072. Triglycerides consist of a three-carbon glycerol backbone covalently linked to three fatty acids. Their fatty acid composition varies in terms of chain length and degree of saturation. Triglyceride molecules are nonpolar and hydrophobic, and are transported in the core of the lipoprotein. Hydrolysis of triglycerides by lipases generates free fatty acids (FFAs) used for energy. Id. Phospholipids, constituents of all cellular membranes, consist of a glycerol molecule linked to two fatty acids. The fatty acids differ in length and in the presence of a single or multiple double bonds. The third carbon of the glycerol moiety carries a phosphate group to which one of four molecules is linked: choline (phosphatidylcholine or lecithin), ethanolamine (phosphatidylethanolamine), serine (phosphatidylserine), or inositol (phosphatidylinositol). Phospholipids, which are polar molecules, more soluble than triglycerides or cholesterol or its esters, participate in signal transduction pathways. Hydrolysis by membrane-associated phospholipases generates second messengers such as diacyl glycerols, lysophospholipids, phoshatidic acids and free fatty acids (FFAs) such as arachidonate that can regulate many cell functions. Id.
The apolipoproteins, which comprise the protein moiety of lipoproteins, vary in size, density in the aqueous environment of plasma, and lipid and apolipoprotein content. The classification of lipoproteins reflects their density in plasma (1.006 gm/mL) as gauged by flotation in the ultracentrifuge. For example, triglyceride-rich lipoproteins consisting of chylomicrons (meaning a class of lipoproteins that transport dietary cholesterol and triglycerides after meals from the small intestine to tissues for degradation) and very low density lipoprotein (VLDL) have a density less than 1.06 gm/mL. Id.
Apolipoproteins have four major roles: (1) assembly and secretion of the lipoprotein (apo B100 and B48); (2) structural integrity of the lipoprotein (apo B, apo E, apo A1, apo AII); (3) coactivators or inhibitors of enzymes (apo A1, C1, CII, CIII); and (4) binding or docking to specific receptors and proteins for cellular uptake of the entire particle or selective uptake of a lipid component (apoA1, B100, E). Id. The role of several apolipoproteins (AIV, AV, D, and J) remain incompletely understood. Id.
Low density lipoprotein (or LDL cholesterol) particles carry cholesterol throughout the body, delivering it to different organs and tissues. The excess keeps circulating in blood. LDL particles contain predominantly cholesteryl esters packaged with the protein moiety apoB 100. Id. at 1076.
High density lipoproteins (or HDL cholesterol) act as cholesterol scavengers, picking up excess cholesterol in the blood and taking it back to the liver where it is broken down. Apolipoprotein A1, the main protein of HDL, is synthesized in the intestine and liver. Lipid-free Apo A1 acquires phospholipids from cell membranes and from redundant phospholipids shed during hydrolysis of triglceride-rich lipoproteins. Lipid-free apo A1 binds to ABCA1 and promotes its phosphorylation via cAMP, which increases the net efflux of phospholipids and cholesterol onto apo A1 to form a nascent HDL particle. Id. These nascent HDL particles will mediate further cellular cholesterol efflux. Id.
The scavenger receptor class B (SR-B1; also named CLA-1 in humans (Id., citing Acton, S. et al, “Identification of scavenger receptor SR-B1 as a high density lipoprotein receptor,” Science 271: 518 (1996)) and the adenosine triphosphate binding cassette transporter A1 (ABCA1) (Id., citing Krinbou, L. et al,” Biogenesis and speciation of nascent apo A1-containing particles in various cell lines,” J. Lipid Res. 46: 1668 (2005)) bind HDL particles. SR-B1, a receptor for HDL (also for LDL and VLDL, but with less affinity), mediates the selective uptake of HDL cholesteryl esters in steroidogenic tissues, hepatocytes and endothelium. ABCA1 mediates cellular phospholipid (and possibly cholesterol) efflux and is necessary and essential for HDL biogenesis. Id.
Cellular cholesterol homeostasis is achieved via at least four major routes: (1) cholesterol de novo biosynthesis from acetyl-CoA in the endoplasmic reticulum; (2) cholesterol uptake by low density lipoprotein (LDL) receptor-mediated endocytosis of LDL-derived cholesterol from plasma; 3) cholesterol efflux mediated by ABC family transporters such as ATP-binding cassette, sub-family A (ABC1), member 1 (ABCA1)/ATP-binding cassette, sub-family G, member 1 (ABCG1), and secretion mediated by apolipoprotein B (ApoB); and (4) cholesterol esterification with fatty acids to cholesterol esters (CE) by acyl-coenzyme A:cholesterol acyltransferase (ACAT) (see FIG. 1 (Jiang, W. and Song, B-L, “Ubiquitin Ligases in Cholesterol Metabolism,” Diabetes Metab. 38: 171-80 (2014)).
Cholesterol Biosynthetic Pathways
Cholesterol synthesis takes place in four stages: (1) condensation of three acetate units to form a six-carbon intermediate, mevalonate; (2) conversion of mevalonate to activated isoprene units; (3) polymerization of six 5-carbon isoprene units to form the 30-carbon linear squalene; and (4) cyclization of squalene to form the steroid nucleus, with a further series of changes to produce cholesterol. (Endo, A., “A historical perspective on the discovery of statins,” Proc. Jpn Acad, Ser. B Phys. Biol. Sci 86(5): 484-93 (2010)).
The mevalonate arm of the cholesterol biosynthesis pathway, which includes enzymatic activity in the mitochondria, peroxisome, cytoplasm and endoplasmic reticulum, starts with the consumption of acetyl-CoA, which occurs in parallel in three cell compartments (the mitochondria, cytoplasm, and peroxisome) and terminates with the production of squalene in the endoplasmic reticulum (Mazein, A. et al., “A comprehensive machine-readable view of the mammalian cholesterol biosynthesis pathway,” Biochemical Pharmacol. 86: 56-66 (2013)). The following are enzymes of the mevalonate arm:
Acetyl-CoA acetyltransferase (ACAT1; ACAT2; acetoacetyl-CoA thiolase; EC 2.3.1.9) catalyzes the reversible condensation of two molecules of acetylcoA and forms acetoacetyl-CoA. Id.
Hydroxymethylglutaryl-CoA synthase (HMGCS1 (cytoplasmic); HMGCS2 (mitochondria and peroxisome); EC 2.3.3.10 catalyzes the formation of 3-hydroxy-3-methylglutaryl CoA (3HMG-CoA) from acetyl CoA and acetoacetyl Co A. Id.
Hydroxymethylglutaryl-CoA lysase (mitochondrial, HMGCL; EC 4.1.3.4) transforms HMG-CoA into Acetyl-CoA and acetoacetate.
3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR; EC 1.1.34) catalyzes the conversion of 3HMG-CoA into mevalonic acid. This step is the committed step in cholesterol formation. HMGCR is highly regulated by signaling pathways, including the SREBP pathway.Id.
Mevalonate kinase (MVK; ATP:mevalonate 5-phosphotransferase; EC 2.7.1.36) catalyzes conversion of mevalonate into phosphomevalonate. Id.
Phosphomevalonate kinase (PMVK; EC 2.7.4.2) catalyzes formation of mevalonate 5-diphosphate from mevalonate 5-phosphate. Id.
Diphosphomevalonate decarboxylase (MVD; mevalonate (diphospho) decarboxylase; EC 4.1.1.33) decarboxylates mevalonate 5-diphosphate, forming isopentenyldiphosphate while hydrolyzing ATP. Id.
Isopentenyl-diphosphate delta-isomerases (ID11; ID12; EC 5.3.3.2) isomerize isopentenyl diphosphate into dimethylallyl diphosphate, the fundamental building blocks of isoprenoids. Id.
Farnesyl diphosphate synthase (FDPS; EC2.5.1.10; EC 2.5.1.1; dimethylallyltranstransferase) catalyzes two reactions that lead to farnesyl diphosphate formation. In the first (EC 2.5.1.1 activity), isopentyl diphosphate and dimethylallyl diphosphate are condensed to form geranyl disphosphate. Next, geranyl diphosphate and isopentenyl diphosphate are condensed to form farnesyl diphosphate (EC 2.5.1.10 activity). Id.
Geranylgeranyl pyrophosphate synthase (GGPS1; EC 1.5.1.29; EC 2.5.1.10; farnesyl diphosphate synthase; EC 2.5.1.1; dimethylallyltranstransferase) catalyzes the two reactions of farnesyl diphosphate formation and the addition of three molecules of isopentenyl diphosphate to dimethylallyl diphosphate to form geranylgeranyl diphosphate. Id.
Farnesyl-diphosphate farnesyltransferase 1 (FDFT1; EC 2.5.1.21; squalene synthase) catalyzes a two-step reductive dimerization of two farnesyl diphosphate molecules (C15) to form squalene (C30). The FDFT1 expression level is regulated by cholesterol status; the human FDFT1 gene has a complex promoter with multiple binding sites for SREBP-1a and SREBP-2. Id.
The sterols arms of the pathway start with Squalene and terminate with cholesterol production on the Bloch and Kandutsch-Russell pathways and with 24 (S),25-epoxycholesterol on the shunt pathway. Id. The following are enzymes of the sterol arms:
Squalene epoxidase (SQLE; EC 1.14.13.132, squalene monooxygenase) catalyzes the conversion of squalene into squalene-2,3-epoxide and the conversion of squalene-2,3-epoxide (2,3-oxidosqualene) into 2,3:22,23-diepoxysqualene (2,3:22,23-dioxidosqualene). The first reaction is the first oxygenation step in the cholesterol biosynthesis pathway. The second is the first step in 24(S),25-epoxycholesterol formation from squalene 2,3-epoxide. Id.
Lanosterol synthase (LSS; OLC; OSC; 2,3-oxidosqualene:lanosterol cyclase; EC 5.4.99.7) catalyzes cyclization of squalene-2,3-epoxide to lanosterol and 2,3:22,23-depoxysqualene to 24(S),25-epoxylanosterol. Id.
Delta(24)-sterol reductase (DHCR24; 24-dehydrocholesterol reductase; EC 1.3.1.72) catalyzes the reduction of the delta-24 double bond of intermediate metabolites. In particular it converts lanosterol into 24, 25-dihydrolanosterol, the initial metabolite of the Kandutsch-Russel pathway and also provides the last step of the Bloch pathway converting desmosterol into cholesterol. Intermediates of the Bloch pathway are converted by DHCR24 into intermediates of the Kandutsch-Russell pathway. Id.
Lanosterol 14-alpha demethylase (CYP51A1; cytochrome P450, family 51, subfamily A, polypeptide 1; EC 1.14.13.70) converts lanosterol into 4,4-dimethyl-5α-cholesta-8,14,24-trien-3β-ol and 24,25-dihydrolanosterol into 4,4-dimethyl-5α-cholesta-8,14-dien-3β-ol in three steps. Id.
Delta (14)-sterol reductase (TM7F2; transmembrane 7 superfamily member 2, EC 1.3.1.70) catalyzes reactions on the three branches of the cholesterol and 24(S),25-epoxycholesterol pathways. Id.
Methylsterol monooxygenase 1 (MSM01; SC4MOL; C-4 methylsterol oxidase; EC 1.14.13.72) catalyzes demethylation of C4 methylsterols. Id.
Sterol-4-alpha-carboxylate 3-dehydrogenase, decarboxylating (NSDHL; NAD(P) dependent steroid dehydrogenase-like; EC 1.1.1.170) participates in several steps of post-squalene cholesterol and 24(S),25-epoxycholeseterol synthesis. Id.
3-keto-steroid reductase (HSD17B7; 17-beta-hydroxysteroid dehydrogenase 7; EC 1.1.1.270) converts zymosterone into zymosterol in the Bloch pathway. Id.
3-Beta-hydroxysteroid-delta(8),delta(7)-isomerase (EBP; emopamil-binding protein; EC5.3.3.5) catalyzes the conversion of delta(8)-sterols into delta(7)-sterols. Id.
Lathosterol oxidase (SC5DL; sterol-C5-desaturase (ERG3 delta-5-desaturase homolog, S. cerevisiae-like; EC 1.14.21.6) catalyzes the production of 7-dehydrocholesterol, 7-dehydrodesmosterol and 24(S),25-epoxy-7-dehydrocholesterol. Id.
7-dehydrocholesterol reductase (DHCR7; EC 1.3.1.21) catalyzes reduction of the C7-C8 double bond of 7-dehydrocholesterol and formation of cholesterol, and produces desmosterol from 7-dehydrodesmosterol and 24(S),25-epoxycholesterol from 24(S),25-epoxy-7-dehydrocholesterol. Id.
Cytochrome P450, family 3, subfamily A, polypeptide 4 (CYP3A4; 1,8-cineole 2-exo-monooxygenase; taurochenodeoxycholate 6α-hydroxylase; EC 1.14.13.97)) catalyzes the hydroxylation of cholesterol leading to 25-hydroxycholesterol and 4β-hydroxycholesterol. Id.
Cholesterol 25-hydroxylase (CH25H; cholesterol 25-monooxygenase; EC 1.14.99.38) uses di-iron cofactors to catalyze the hydroxylation of cholesterol to produce 25-hydroxycholesterol, and has the capacity to catalyze the transition of 24-hydroxycholesterol to 24, 25-dihydroxycholesterol. Id.
Cytochrome P450, family 7, subfamily A, polypeptide 1 (CYP7A1; cholesterol 7-alpha-hydroxylase; EC 1.14.13.17) is responsible for introducing a hydrophilic moiety at position 7 of cholesterol to form 7α-hydroxycholesterol. Id.
Cytochrome P450, family 27, subfamily A, polypeptide 1 (CYP27A1; Sterol 27-hydroxylase; EC 1.14.13.15) catalyzes the transition of mitochondrial cholesterol to 27-hydroxycholesterol and 25-hydroxycholesterol. Id.
Cytochrome P450 46A1 (CYP46A1, cholesterol 24-hydroxylase, EC 1.14.13.98) catalyzes transformation of cholesterol into 24(S)-hydroxycholesterol. Id.
Intermediates in Cholesterol Synthesis as Physiological Regulators
Intermediates in cholesterol synthesis, mostly sterols (e.g. 7-dehydrocholesterol, which is converted to cholesterol by DHCR7 (7-dehydrocholesterol reductase), but which also is a precursor for vitamin D), have been credited with having regulatory functions distinct from those of cholesterol. (Sharpe, L J and Brown, A J, “Controlling cholesterol synthesis beyond 3-hydroxy-3-methylglutaryl CoA reductase (HMGCR),” J. Biol. Chem. 288 (26): 18707-715 (2013)).
C4-methylsterols are produced by lanosterol 14α-demethylase (encoded by CYP51A1 (cytochrome P450, family 51, subfamily A, polypeptide 1) and demethylated by SC4MOL (sterol-C4-methyl oxidase like 1; methylsterol monooxygenase 1) and its partner, NSDHL (NAD(P)-dependent steroid dehydrogenase-like; sterol-4-α-carboxylate 3-dehydrogenase, decarboxylating).Id.
24, 25-dihydrolanosterol purportedly is the primary degradation signal for 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) (Id., citing Song, B L, et al, “Insig-mediated degradation of HMG-CoA reductase stimulated by lanosterol, an intermediate in the synthesis of choleseterol,” Cell Meta. 1: 179-89 (2005); Lange, Y. et al, “Effectors of rapid homeostatic responses of endoplasmic reticulum cholesterol and 3-hydroxy-3-methylglutaryl-CoA reductase,” J. Biol. Chem. 283: 1445-55 (2008)).
The nonsterol intermediate squalene has been implicated in stimulating HMGCR degradation (Id., citing Leichner, G S, et al, “Metabolically regulated endoplasmic reticulum-associated degradation of 3-hydroxy-3-methylglutaryl-CoA reductase. Evidence for requirement of a geranylgeranylated protein,” J. Biol. Chem. 286: 32150-61 (2011)).
A number of cholesterol synthesis intermediates can serve as activating ligands of the nuclear liver X receptor (LXR), which up-regulates cholesterol export genes and represses inflammatory genes. These sterols include 24,25-dihydrolanosterol (Id., citing Zhu, J. et al, “Effects of FoxO4 overexpression on cholesterol biosynthesis, triacylglycerol accumulation, and glucose uptake,” J. Lipid Res. 51: 1312-24 (2010)), meiosis-activating sterols (MASs) (Id., citing He, M, et al, “Mutations in the human SC4MOL gene encoding a methyl sterol oxidase cause psoriasiform dermatitis, microcephaly, and developmental delay,” J. Clin. Invest. 121: 97 6-984 (2011)) and desmosterol (Id., citing Yang, C. et al, “Sterol intermediates from choleseterol biosynthetic pathway as liver X receptor ligands,” J. Biol. Chem. 281: 27816-826 (2006); Spann, N J et al, “Regulated accumulation of desmosterol integrates macrophage lipid metabolism and inflammatory responses,” Cell 151: 138-52 (2012)).
The oxysterol 24(S),25-epoxycholesterol (24,25-EC), a potent LXR agonist (Id., citing Lehmann, J M et al, “Activation of the nuclear receptor LXR by oxysterols defines a new hormone response pathway,” J. Biol. Chem. 272: 3137-40 (1997)), is produced in a shunt pathway in sterol synthesis (Id., citing Spencer, T A, et al, “24(S),25-epoxyscholesterol. Evidence consistent with a role in the regulation of hepatic cholestrogenesis,” J. Biol. Chem. 260: 13391-94 (1985)), and its production is determined by the relative activities of squalene monooxygenase (SM) and lanosterol synthase (LS). Partial inhibition or knockdown of LS diverts more flux into the shunt pathway, producing more 14,15-epoxycholesterol (14,15-EC) (Id., citing Dang, H. et al, “Suppression of 2,3-oxidosqualene cyclase by high fat diet contributes to liver X receptor-α-mediated improvement of hepatic lipid profile,” J. Biol. Chem. 284: 6218-26 (2009)), whereas overexpression of LS abolishes 24,25-EC production (Id., citing Wong, J. et al, “Endogenous 24(S),25-epoxycholesterol fine-tunes acute control of cellular cholesterol homeostasis,” J. Biol. Chem. 283: 700-707 (2008)). Conversely, overexpression of SM increases 24,25-EC production (Id., citing Zerenturk, E J et al, “The endogenous regulator 24(S),25-epoxycholesterol inhibits cholesterol synthesis at DHCR24 (Seladin-1),” Biochim. Biophys. Acta 1821: 1269-77 (2012)). The extent to which SM and LS are differentially regulated to alter 14,15-EC production is not known.
Cholesterol Uptake by Low Density Lipoprotein (LDL) Receptor-mediated Endocytosis of LDL-derived Cholesterol from Plasma
The LDL receptor regulates the entry of cholesterol into cells; tight control mechanisms alter its expression on the cell surface, depending on need. Braunwald's Heart Disease, P. Libby, R. Bonow, D. Mann and D. Zipes, Eds., 8th Edition, Saunders Elsevier, Philadelphia, Pa. (2008) at 1072. Other receptors for lipoproteins include several that bind VLDL, but not LDL. Id. The LDL receptor-related peptide, which mediates the uptake of chylomicron remnants and VLDL, preferentially recognizes apolipoprotein E (apo E) (Id., citing Hiltunen, T P et al, Expression of LDL receptor, VLDL receptor, LDL receptor-related protein, and scavenger receptor in rabbit atherosclerotic lesions: Marked induction of scavenger receptor and VLDL receptor expression during lesion development,” Circulation 97: 1079 (1998)). The LDL receptor-related peptide interacts with hepatic lipase. A specific VLDL receptor also exists (Id., citing Nimph, J, and Schneider, W J, “The VLDL receptor: an LDL receptor relative with eight ligand binding repeats, LR8. Atherosclerosis 141: 191-202 (1998)). The interaction between hepatocytes and the various lipoproteins containing apo E is complex and involves cell surface proteoglycans that provide a scaffolding for lipolytic enzymes (lipoprotein lipase and hepatic lipase) involved in remnant lipoprotein recognition (Id., citing Mahley, R W, Ji, Zs, “Remnant lipoprotein metabolism: key pathways involving cell-surface heparin sulfate proteoglycans and apolipoprotein E,” J. Lipid Res. 40: 1-(1999); Barown M I et al, “A macrophage receptor for apolipoprotein B48: cloning, expression and atherosclerosis, Proc. Natl Acad. Sci. USA 97: 7488 (2000); de Man, F H et al, “Lipolysis of very low density lipoproteins by heparin sulfate proteoglycan-bound lipoprotein lipase,” J. Lipid Res. 38: 2465 (1997)).
Macrophages express receptors that bind modified (especially oxidized) lipoproteins. These scavenger lipoprotein receptors mediate the uptake of oxidized LDL into macrophages. In contrast to the regulated LDL receptor, high cellular cholesterol content does not suppress scavenger receptors, enabling the intimal macrophages to accumulate abundant cholesterol, become foam cells, and form fatty streaks. Endothelial cells also can take up modified lipoproteins through a specific receptor, such as Lox-1 (Sawamura, T. et al, “an endothelial receptor for oxidized low-density lipoprotein,” Nature 386: 73 (1997)).
Cholesterol Efflux is Mediated by ABC Family Transporters Such as ATP-binding cassette, Sub-family a (ABC1), Member 1 (ABCA1)/ATP-binding Cassette, Sub-family G, Member 1 (ABCG1), and Secretion Mediated by Apolipoprotein B (ApoB);
Because most cells in the body do not express pathways for catabolizing cholesterol, efflux of cholesterol is critical for maintaining homeostasis. (Phillips, M C, “Molecular Mechanisms of Cellular Cholesterol Efflux,” J. Biol. Chem. 289 (35): 24020-29 (2014)). High density lipoprotein (HDL) comprises a heterogeneous population of microemulsion particles 7-12 nm in diameter containing a core of cholesterol ester (CE) and triglyceride (TG) molecules stabilized by a monomolecular layer of phospholipid (PL) and apolipoprotein (apo), of which apo1 is the principal component (Id. citing Phillips, M C, “New insights into the determination of HDL structure by apolipoproteins,” J. Lipid Res. 54: 2034-48 (2013)). The presence of PL in the particles enables HDL to solubilize and transport unesterified (free) cholesterol (FC) released from cells, thereby mediating removal of cholesterol from cholesterol-loaded arterial macrophages and transport to the liver for catabolism and elimination from the body (“reverse cholesterol transport”) (Id., citing Rothblat, G H and Phillips, M C, “High-density lipoprotein heterogeneity and function in reverse cholesterol transport,” Curr. Opin. Lipidol. 21: 229-38 (2010); Rosenson, R S et al, “Cholesterol efflux and atheroprotection: advancing the concept of reverse cholesterol transport,” Circulation 125: 1905-19 (2012)).
The first step in reverse cholesterol transport is efflux of FC from the cell plasma membrane to HDL. Id. In the case of macrophages, four efflux pathways have been identified: the aqueous diffusion efflux pathway, the scavenger receptor class B, type 1 (SR-B1) pathway; the ATP binding cassette transporter G1 (ABCG1) pathway and the ATP-binding cassette transporter A1 (ABCA1) pathway. Id. The first two processes, which are passive, involve simple diffusion (aqueous diffusion pathway) and facilitated diffusion (SR-B1-mediated pathway). Id. The two active processes involve members of the ATP-binding cassette (ABC) family of transmembrane transporters, namely ABCA1 and ABCG1. Id. The efficiency of an individual serum sample in accepting cellular cholesterol depends upon both the distribution of HDL particles present and the levels of cholesterol transporters expressed in the donor cells. Id.
Aqueous Diffusion Efflux Pathway
HDL is the component of serum responsible for mediating FC efflux from monolayers of mouse L-cell fibroblasts. Id. Transfer occurs by an aqueous phase intermediate where monomeric FC molecules desorb from a donor particle and diffuse until they are absorbed by an acceptor particle. The rate of transfer of the highly hydrophobic cholesterol molecule from donor to acceptor is limited by the rate of desorption into the aqueous phase, which is sensitive to the physical state of the phospholipid (PL) milieu in which the transferring FC molecules are located. The net mass FC efflux from cells to HDL in the extracellular medium is promoted by metabolic trapping in which return of released FC to the cell is prevented by esterification, when lecithin-cholesterol aceyltransferase acts on HDL (Id., citing Czarnecka, H. and Yokoyama, S., “Regulation of cellular cholesterol efflux by lecithin: cholesterol acyltransferase reaction through nonspecific lipid exchange,” J. Biol. Chem. 271: 1023-27 (1996)).
SR-B1 Efflux Pathway
SR-B1 is a member of the CD36 superfamily of scavenger receptor proteins that also includes lysosomal integral membrane protein-2 (LIMP-2). Id. The receptor is most abundantly expressed in liver, where it functions in the reverse cholesterol transport pathway and in steroidogenic tissue, where it mediates cholesterol delivery (Id., citing Zannis, V. et al, “Role of apoA-1, ABCA1, LCAT and SR-B1 in the biogenesis of HDL,” J. Mol. Med. 84: 276-94 (2006)). It is a homo-oligomeric glycoprotein located in the plasma membrane with two N- and C-terminal transmembrane domains and a large central extracellular domain (Id., citing Williams, D L, et al, “Scavenger receptor B1 and cholesterol trafficking,” Curr. Opin. Lipidol. 10: 329-39 (1999); Meyer, J M et al, “New developments in selective cholesteryl ester uptake,” Curr. Opin. Lipidol. 24: 386-92 (2013)). In 1996, it was established that SR-B1 is an HDL receptor that mediates cholesterol uptake into cells. This process involves selective transfer of the cholesterol ester (CE) in an HDL particle into the cell without endocytic uptake and degradation of the HDL particle itself. In addition to promoting delivery of HDL cholesterol to cells, SR-B1 also enhances efflux of cellular cholesterol to HDL (Id., citing Ji, Y et al, “Scavenger receptor B1 promotes high density lipoprotein-mediated cellular cholesterol efflux,” J. Biol. Chem. 272: 20982-985 (1997); Jian, B. et al, “Scavenger receptor class B type 1 as a mediator of cellular cholesterol efflux to lipoproteins and phospholipid acceptors,” J. Biol. Chem. 273: 5599-5606 (1998)) with the two processes being related (Id., citing Gu, X et al, “Scavenger receptor class B, type 1-mediated [3H]cholesterol efflux to high and a low density lipoproteins is dependent on lipoprotein binding to the receptor,” J. Biol. Chem. 275: 29993-30001 (2000)). For CE selective uptake via SR-B1, HDL binding and CE uptake are tightly coupled. The mechanism for CE uptake from HDL involves a two-step process in which HDL first binds to the receptor and then CE molecules transfer from the bound HDL particle into the cell plasma membrane, with enhanced binding of larger HDL particles to SR-B1 increasing the selective delivery of CE (Id., citing Thuahnai, S T, et al, “SR-B1-mediated cholesteryl ester selective uptake and efflux of unesterified cholesterol: influence of HDL size and structure,”” J. Biol. Chem. 279: 12448-455 (2004)). The binding of HDL to the extracellular domain of SR-B1 involves direct protein-protein contact with a recognition motif being the amphipathic a helix characteristic of HDL apolipoproteins (Id., citing Williams, D L et al, “Binding and cross-linking studies show that scavenger receptor B1 interacts with multiple sites in apolipoprotein A-1 and identify the class A amphipathic a helix as a recognition motif,” J. Biol. Chem. 275: 18897-18904 (2000). Consistent with CE selective uptake being a passive process, the rate of uptake is proportional to the amount of CE initially present in the HDL particles.
FC efflux and HDL binding are not completely coupled, and the FC efflux mechanism proceeds by different pathways at low and high extracellular HDL concentrations (Id., citing Thuahnai, S T, et al, “SR-B1-mediated cholesteryl ester selective uptake and efflux of unesterified cholesterol: influence of HDL size and structure,”” J. Biol. Chem. 279: 12448-455 (2004); de la Llera-Moya, M. et al, “Scavenger receptor B1 (SR-B1) mediates free cholesterol flux independently of HDL tethering to the cell surface,” J. Lipid Res. 40: 575-80 (1999)). At low HDL concentrations, binding of HDL to SR-B1 is critical, allowing bidirectional FC transit through the hydrophobic tunnel present in the extracellular domain of the receptor. Because the FC concentration gradient between the bound HDL particle and the cell plasma membrane is opposite to that of CE, the relatively high FC/PL ratio in the plasma membrane causes the direction of net mass FC transport to be out of the cell. Consistent with this concept, enhancing the PL content of HDL promotes FC efflux from cells (Id., citing Yancey, P G, et al, “High density lipoprotein phospholipid composition is a major determinant of the bi-directional flux and net movement of cellular free cholesterol mediated by scavenger receptor B1,” J. Biol. Chem. 275: 36596-36604 (2000)). Larger HDL particles promote more FC efflux than smaller HDL, because they bind better to SR-B1 (Id., citing Thuahnai, S T, et al, “SR-B1-mediated cholesteryl ester selective uptake and efflux of unesterified cholesterol: influence of HDL size and structure,”” J. Biol. Chem. 279: 12448-455 (2004)). At higher HDL concentrations where binding to the receptor is saturated, FC efflux still increases with increasing HDL concentration (Id., citing Thuahnai, S T, et al, “SR-B1-mediated cholesteryl ester selective uptake and efflux of unesterified cholesterol: influence of HDL size and structure,”” J. Biol. Chem. 279: 12448-455 (2004)), because SR-B1 induces reorganization of the FC in the cell plasma membrane.
ABCG1 Efflux Pathway
ABCG1 functions as a homodimer, and is expressed in several types, where it mediates cholesterol transport through its ability to translocate cholesterol and oxysterols across membranes. Id. Expression of ABCG1 enhances FC and PL efflux to HDL (Id., citing Wang, N. et al, “ATP-binding cassette transporters G1 and G4 mediate cellular cholesterol efflux to high-density lipoproteins,” Proc. Natl Acad. Sci. USA 101: 9774-79 (2004); Kennedy, M A et al, “ABCG1 has a critical role in mediating cholesterol efflux to HDL and preventing cellular lipid accumulation,” Cell Metab. 1: 121-31 (2005)), but not to lipid-free apoA-1 (Id., citing Vaughan, A M and Oram, J F, “ABCG1 redistributes cell cholesterol to domains removable by high density lipoprotein but not by lipid-depleted apolipoproteins,” J. Biol. Chem. 280: 20150-57 (2005); Sankaranarayanan, S. et al., “Effects of acceptor composition and mechanism of ABCG1-mediated cellular free cholesterol efflux,” J. Lipid Res. 50: 275-84 (2009)). The presence of the transporter induces reorganization of plasma membrane cholesterol so that it becomes accessible to cholesterol oxidase (Id., citing Vaughan, A M and Oram, J F, “ABCG1 redistributes cell cholesterol to domains removable by high density lipoprotein but not by lipid-depleted apolipoproteins,” J. Biol. Chem. 280: 20150-57 (2005)), creating an activated pool of plasma membrane FC, and desorption of FC molecules from this environment into the extracellular medium is facilitated. Increased expression of ABCG1 enhances FC efflux to HDL2 and HDL3 similarly, but has no effect on the influx of FC from these lipoprotein particles.
ABCA1 Efflux Pathway
ABCA1 is a full transporter whose expression is up-regulated by cholesterol loading, which leads to enhanced FC efflux. Id. Binding and hydrolysis of ATP by the two cytoplasmic, nucleotide-binding domains control the conformation of the transmembrane domains so that the extrusion pocket is available to translocate substrate from the cytoplasmic leaflet to the exofacial leaflet of the bilayer membrane. Id. ABCA1 actively transports phosphatidylcholine, phosphatidylserine, and sphingomyelin with a preference for phosphatidylcholine (Id., citing Quazi, F and Molday, R S, “Differential phospholipid substrates and directional transport by ATP-binding cassette proteins ABCA, ABCA7, and ABCA4 and disease-causing mutants,” J. Biol. Chem. 288: 34414-26 (2013)). This PL translocase activity leads to the simultaneous efflux of PL and FC (Id., citing Gillotte, K L, et al, “Removal of cellular cholesterol by pre-β-HDL involves plasma membrane microsolubilization,” J. Lipid Res. 39: 1918-28 (1998); Smith, J D et al, “ABCA1 mediates concurrent cholesterol and phospholipid efflux to apolipoprotein A-1,” J. Lipid Res. 45: 635-44 (2004)) to lipid-free apoA-1 (plasma pre-β1-HDL). The cellular FC released to apoA-1 originates from both the plasma membrane and the endosomal compartment (Id., citing Chen, W. et al, “Preferential ATP-binding cassette transporter A1-mediated cholesterol efflux from late endosomes/lysosomes,” J. Biol. Chem. 276: 43564-69 (2001)).
The PL translocase activity of ABCA1 induces reorganization of lipid domains in the plasma membrane (Id., citing Landry, Y D, et al, “ATP-binding cassette transporter A1 expression disrupts raft membrane microdomains through its ATPase-related functions,” J. Biol. Chem. 281: 36091-101 (2006)). ABCA1 exports PL and FC to various plasma apolipoproteins. Besides FC efflux, intracellular signaling pathways are activated by the interaction of apoA-1 with ABCA1 (Id., citing Mineo, C. and Shaul, P W, “Regulation of signal transduction by HDL,” J. Lipid Res. 54: 2315-24 (2013); Liu, Y, and Tang, C., “Regulation of ABCA1 functions by signaling pathways,” Biochim. Biophys. Acta, 1821: 522-29 (2012)).
It is well established that the activity of ABCA1 in the plasma membrane enhances binding of apoA-1 to the cell surface, but there has been controversy about the role of this binding in the acquisition of membrane PL by apo-A1. Id. It has been proposed that apoA-1 acquires PL either directly from ABCA1 while it is bound to the transporter, or indirectly at a membrane lipid-binding site created by ABCA1 activity. Id.
The ABCA1-mediated assembly of nascent HDL particles occurs primarily at the cell surface (Id., citing Faulkner, L E, et al, “An analysis of the role of a retroendocytosis pathway in ABCA1-mediated cholesterol efflux from macrophages,” J. Lipid Res. 49: 1322-32 (2008); Denis, M. et al, “ATP-binding cassette A-1-mediated lipidation of apolipoprotein A-1 occurs at the plasma membrane and not in the endocytic compartments,” J. Biol. Chem. 283: 16178-186 (2008)), where extracellular apoA-1 for HDL particle formation is available. The FC/PL ratio in nascent HDL particles created by ABCA1 activity is dependent upon the cell type and metabolic status of the cell, but the population of larger particles is always relatively FC-rich as compared with the smaller particles.
Regulation of cholesterol efflux depends in part on the ABCA1 pathway, controlled in turn by hydroxysterols (especially 24 and 27-OH cholesterol, which act as ligands for the liver-specific receptor (LXR) family of transcriptional regulatory factors. Braunwald's Heart Disease, P. Libby, R. Bonow, D. Mann and D. Zipes, Eds., 8th Edition, Saunders Elsevier, Philadelphia, Pa. (2008) at 1076.
Cholesterol Esterification with Fatty Acids to Cholesterol Esters (CE) by Acyl-coenzyme A:cholesterol Acyltransferase (ACAT)
Cholesterol content in membranes regulates the cholesterol acyltransferase (CAT) pathway at the level of protein regulation. (Braunwald's Heart Disease, P. Libby, R. Bonow, D. Mann and D. Zipes, Eds., 8th Edition, Saunders Elsevier, Philadelphia, Pa. (2008) at 1076, citing Willner, E. et al, “Deficiency of acyl CoA:cholesterol aceyltransferase 2 prevents atherosclerosis in apolipoprotein E-deficient mice., Proc. Natl Acad. Sci. USA 100: 1262 (2003). Humans express two separate forms of ACAT (ACT1 and ACAT2), which derive from different genes and mediate cholesterol esterification in cytoplasm and in the endoplasmic reticulum lumen for lipoprotein assembly and secretion.
Regulation of Cholesterol Content
Under conditions of cell cholesterol sufficiency, the cell can decrease its input of cholesterol by decreasing the de novo synthesis of cholesterol. The cell can also decrease the amount of cholesterol that enters the cell via the LDL-R, increase the amount stored as cholesteryl esters, and promote the removal of cholesterol by increasing its movement to the plasma membrane for efflux.
The regulation of HMG CoA reductase, the rate limiting step in cholesterol biosynthesis, has been investigated in detail. However, this enzyme acts very early in the cholesterol synthesis pathway. There is accumulating evidence that enzymes beyond HMG CoA reductase serve as flux controlling points, and that regulation of cholesterol synthesis can occur at multiple levels throughout the pathway. (Sharpe, L J and Brown, A J, “Controlling cholesterol synthesis beyond 3-hydroxy-3-methylglutaryl CoA reductase (HMGCR),” J. Biol. Chem. 288 (26): 18707-715 (2013)).
Transcriptional Regulation
Sterol Regulatory Element-binding Proteins (SREBPs)
SREBPs, membrane bound transcription factors that coordinate the synthesis of fatty acids and cholesterol, the two major building blocks of membranes (Brown, M S & Goldstein, J L, “The SREBP pathway: regulation of cholesterol metabolism by proteolysis of a membrane-bound transcription factor,” Cell 89: 331-40 (1997)), belong to the basic helix-loop-helix-leucine zipper (bHLH-Zip) family of transcription factors. There are three SREBP proteins (SREB-1a, SREBP-1c, and SREBP-2) from two srebp genes designated srebp1 and srebp2. Id. The SREBP2 isoform plays a major role in regulating cholesterol synthetic genes.
As shown in Table 1, nearly all of the genes encoding cholesterol synthesis enzymes are SREBP targets.
TABLE 1Genes Encoding Cholesterol Syhnthesis Enzymes that are SREBP TargetsGene NameGene SymbolSREBP TargetAcetyl-CoA acetyltransferase,ACAT2Yescytosolic3-hydroxy-3-methylglutaryl-MHGCS1YesCoA synthase 1 (soluble)3-hydroxy-3-methylglutaryl-HMGCRYesCoA reductaseMevalonate kinaseMVKYesPhosphomevalonate kinasePMVKYesMevalonateMVDYes(diphospho)decarboxylaseIsopentenyl-diphosphate Δ-ID11/ID12Yesisomerase ½Farnesyl-diphosphate synthaseFDFSYesGeranylgeranyl-diphosphateGGPS1Yessynthase 1Farnesyl-diphosphateFDFT1Yesfarnesyltransferase 1Squalene epoxidaseSQLEYesLanosterol synthase (2,3-LSSYesoxidosqualene-lanosterolcyclase)Cytochrome P450, family 51, CYPS1A1Yessubfamily A, polypeptide 1Transmembrane 7 superfamilyTM75F2Yesmember 2Lamin B receptorLBRNoMethylsterol monooxygenaseSCAMOLYes1NAD(P)-dependent steroidNSDHLYesdehydrogenase-likeHydroxysteroid 17β-HSD17B7Yesdehydrogenase 7Emopamil-binding proteinEBPYes(sterol isomerase)Sterol C5-desaturaseSC5DYes7-DehydrocholesterolDHCR7Yesreductase24 DehydrocholesterolDHCR24YesreductaseTaken from Sharpe, L J and Brown, A J, “Controlling cholesterol synthesis beyond 3-hydroxy-3-methylglutaryl CoA reductase (HMGCR),” J. Biol. Chem. 288 (26): 18707-715 (2013))
SREBPs coordinately regulate the cholesterol biosynthetic pathway and receptor-mediated endocytosis of LDL at the level of gene transcription. (Brown, M S and Goldstein, J L, “A proteolytic pathway that controls the cholesterol content of membranes, cells and blood,”Proc. Natl Acad. Sci. USA 96: 11041-48 (1999)). In the cholesterol biosynthetic pathway, SREBPs regulate transcription of HMG CoA reductase as well as transcription of genes encoding many other enzymes in the cholesterol biosynthetic pathway, including HMG CoA synthase, farnesyl diphosphate synthase and squalene synthase. Id. Studies investigating regulation of the DHCR24 promoter provided evidence of binding sites for SREBP-2 [Daimiel, L A, et al, “Promoter analysis of the DHCR24 (3β-hydroxysterol Δ24-reductase) gene: characterization of SREBP (sterol-regulatory element-binding-protein)-mediated activation,” Biosci. Rep. (2013)/art:e000/doi 10.1042/BSR20120095); Zerenturk, E J, et al, “Sterols regulate 3β-hydroxysterolΔ24-reductase (DHCR24) via dual sterol regulatory elements: cooperative induction of key enzymes in lipid synthesis by sterol regulatory element binding proteins,” Biochim. Et Biophys. Acta 1821 (10): 1350-60 (2012)). The SREBPs also regulate the LDL receptor, which supplies cholesterol through receptor mediated endocytosis, and modulate transcription of genes encoding enzymes of fatty acid synthesis and uptake, including acetyl CoA carboxylase, fatty acid synthase, stearoyl CoA desaturase-1 and lipoprotein lipase. (Brown, M S & Goldstein, J L, “The SREBP pathway: regulation of cholesterol metabolism by proteolysis of a membrane-bound transcription factor,” Cell 89: 331-40 (1997)).
Nascent SREBPs are targeted to the endoplasmic reticulum (ER) membrane without any transcription activity, because they are not available for their target genes, which are located in the nucleus. (Brown, M S & Goldstein, J L, “The SREBP pathway: regulation of cholesterol metabolism by proteolysis of a membrane-bound transcription factor,” Cell 89: 331-40 (1997)). To enhance transcription when cellular sterol is low, the active NH2-terminal domains of SREBPs are released from endoplasmic reticulum membranes by two sequential cleavages that must occur in the proper order. The first is catalyzed by Site-1 protease (S1P), a membrane bound subtilisin-related serine protease that cleaves the hydrophilic loop of SREBP that projects into the endoplasmic reticulum lumen. (Brown, M S and Goldstein, J L, “A proteolytic pathway that controls the cholesterol content of membranes, cells and blood,” Proc. Natl Acad. Sci. USA 96: 11041-48 (1999)). The second cleavage, at Site-2, requires the action of S2P, a hydrophobic protein that appears to be a zinc metalloprotease, and takes place within a membrane-spanning domain of SREBP. Id. Sterols block SREBP processing by inhibiting S1P. Id. Sterols block the proteolytic release process by selectively inhibiting cleavage by S1P; S2P is regulated indirectly because it cannot act until SREBP has been processed by S1P. Id.
SREBP cleavage-activating protein (SCAP), an integral ER membrane regulatory protein, is required for cleavage at Site 1 and is the target for sterol suppression of this cleavage, i.e., SCAP loses its activity when sterols overaccumulate in cells. Id. Within cells, SCAP is found in a tight complex with SREBPs. Id. SCAP contains two distinct domains: a hydrophobic N-terminal domain that spans the membrane eight times and a hydrophilic C-terminal domain that projects into the cytosol. (DeBose-Boyd, R. A. “Feedback Regulation of Cholesterol Synthesis: Sterol-accelerated ubiquitination and degradation of HMG CoA Reductase,” Cell Res. 18 (6): 609-21 (2008)) A 160 amino acid segment of the membrane domain of SCAP has been termed the sterol-sensing domain. (Brown, M S and Goldstein, J L, “A proteolytic pathway that controls the cholesterol content of membranes, cells and blood,” Proc. Natl Acad. Sci. USA 96: 11041-48 (1999)). The C-terminal domain of SCAP mediates a constitutive association with SREBPs, which is required for SCAP-dependent translocation of SREBPs from the ER to Golgi in sterol-deprived cells. (DeBose-Boyd, R. A. “Feedback Regulation of Cholesterol Synthesis: Sterol-accelerated ubiquitination and degradation of HMG CoA Reductase,” Cell Res. 18 (6): 609-21 (2008)). The NH2-terminal bHL-Zip domain with full transcription activity is released from the membrane to reach the nucleus and act as a transcription factor to activate genes responsible for cholesterol and fatty acid biosynthesis and LDL uptake (Brown, M S and Goldstein, J L, “A proteolytic pathway that controls the cholesterol content of membranes, cells and blood,”Proc. Natl Acad. Sci. USA 96: 11041-48 (1999)).
When sterols build up within cells, the proteolytic release of SREBPs from ER membranes is blocked, the NH2-terminal domains that have already entered the nucleus are rapidly degraded, and, as a result, transcription of all of the target genes declines. (Id). This decline is complete for the cholesterol biosynthetic enzymes whose transcription is entirely dependent on SREBPs, but less complete for the fatty acid biosynthetic enzymes whose basal transcription can be maintained by other factors.
Other Factors
Besides SREBP, numerous other transcription factors have been implicated in the transcriptional control of the various enzymes in cholesterol biosynthesis. (Sharpe, L J and Brown, A J, “Controlling cholesterol synthesis beyond 3-hydroxy-3-methylglutaryl CoA reductase (HMGCR),” J. Biol. Chem. 288 (26): 18707-715 (2013)).
Liver X Receptors (LXRs)
Liver X receptors (LXRs) are ligand-activated transcription factors of the nuclear receptor superfamily. (Baranowski, M., “Biological role of liver X receptors,” J. Physiol. Pharmacology. 59 Suppl. 7: 31-55 (2008)). There are two LXR isoforms (termed alpha and beta), which, upon activation, form heterodimers with retinoid X receptor and bind to LXR response elements found in the promoter region of the target genes. Id. High expression levels of LXRα in metabolically active tissues fit with a central role of the receptor in lipid metabolism, while LXRβ is more ubiquitously expressed. (Pehkonen, P. et al., “Genome-wide landscape of liver X receptor chromatin binding and gene regulation in human macrophages,” BMC Genomics 13: 50 (2012)). Both LXRs are found in various cells of the immune system, such as macrophages, dendritic cells and lymphocytes. Id. In macrophages, the accumulation of excess lipoprotein-derived cholesterol activates LXR and triggers the induction of a transcriptional program for cholesterol efflux, such as ATP-binding cassette transporter (ABC) A1 (ABCA1) and ABCG1, while in parallel the receptor transrepresses inflammatory genes, such as inducible nitric oxide synthase, interleukin 1β, and monocyte chemotactic protein-1. Id. LXR has been reported to regulate cholesterol biosynthesis by directly silencing the gene expression of two cholesterogenic enzymes (FDFT1 and CYP51A1). (Sharpe, L J and Brown, A J, “Controlling cholesterol synthesis beyond 3-hydroxy-3-methylglutaryl CoA reductase (HMGCR),” J. Biol. Chem. 288 (26): 18707-715 (2013), citing Wang, Y. et al, “Regulation of cholesterologenesis by the oxysterol receptor, LXRα,” J. Biol. Chem. 283: 26332-339 (2008)).
Endogenous agonists of the LXRs include oxysterols, which are oxidized cholesterol derivatives. (Baranowski, M., “Biological role of liver X receptors,” J. Physiol. Pharmacol. 59 Suppl. 7: 31-55 (2008)). LXRs have been characterized as key transcriptional regulators of lipid and carbohydrate metabolism, and were shown to function as sterol sensors protecting the cells from cholesterol overload by stimulating reverse cholesterol transport and activating its conversion to bile acids in the liver. Id. This finding led to identification of LXR agonists as potent anti-atherogenic agents in rodent models of atherosclerosis. Id. However, first-generation LXR activators were also shown to stimulate lipogenesis via SREBP1c leading to liver steatosis and hypertriglyceridemia. Id.
Despite their lipogenic action, LXR agonists possess antidiabetic properties. Id. LXR activation normalizes glycemia and improves insulin sensitivity in rodent models of type 2 diabetes and insulin resistance. Id. Although antidiabetic action of LXR agonists is thought to result predominantly from suppression of hepatic gluconeogenesis, some studies suggest that LXR activation may also enhance peripheral glucose uptake. Id.
Published reports of anti-proliferative effects of synthetic LXR ligands on breast, prostate, ovarian, lung, skin, and colorectal cancer cells suggest that LXRs are potential targets in cancer prevention and treatment. Nguyen-Vu, T. et al, “Liver x receptor ligands disrupt breast cancer cell proliferation through an E2F-mediated mechanism,” Breast Cancer Res. 15: R51 (2013). Cell line-specific transcriptional responses and a set of common responsive genes were shown by microarray analysis of gene expression in four breast cell lines [MCF-7 (ER+), T-47D (ER+), SK-BR-3 (ER−), and MDA-MB-231] following treatment with the synthetic LXR ligand GW3965. Id. In the common responsive gene set, upregulated genes tend to function in the known metabolic effects of LXR ligands and LXRs whereas the downregulated genes mostly include those which function in cell cycle regulation, DNA replication, and other cell proliferation-related processes. Id. Transcription factor binding site analysis of the downregulated genes revealed an enrichment of E2F binding site sequence motifs. Id. Correspondingly, E2F2 transcript levels are downregulated following LXR ligand treatment. Id. Knockdown of E2F2 expression, similar to LXR ligand treatment, resulted in a significant disruption of estrogen receptor positive breast cancer cell proliferation. Id. Ligand treatment also decreased E2F2 binding to cis-regulatory regions of target genes.
Expression of activated LXRα blocks proliferation of human colorectal cancer cells and slows the growth of xenograft tumors in mice, and reduces intestinal tumor formation after administration of chemical carcinogens in Apc(min/+) mice. Lo Sasso, G. et al., “Liver X receptors inhibit proliferation of human colorectal cancer cells and growth of intestinal tumors in mice,” Gastroenterology 144(7): 1497-507 (2013). A link of LXRs to apoptosis has been reported. (Pehkonen, P. et al, “Genome-wide landscape of liver X receptor chromatin binding and gene regulation in human macrophages,” BMC Genomics 13: 50 (2012)).
MicroRNAs and Alternative Splicing
Overall, relatively little has been reported on miRNAs in the context of cholesterol synthesis. (Sharpe, L J and Brown, A J, “Controlling cholesterol synthesis beyond 3-hydroxy-3-methylglutaryl CoA reductase (HMGCR),” J. Biol. Chem. 288 (26): 18707-715 (2013)) In the context of cholesterol metabolism, perhaps the best studied microRNA (miRNA) is miR-33, an intronic miRNA encoded in the SREBP genes that controls cellular cholesterol export, whereas its SREBP host genes stimulate cholesterol synthesis (Id., citing Fernandez-Hernando, et al, “MicroRNAs in metabolic disease,” Arterioscl. Thromb. Vasc. Biol. 33: 178-85 (2013)).
Alternative splicing of HMGCR is regulated by sterols, with proportionally less of an unproductive transcript present when sterol levels are low and more when sterol levels are higher (Id., citing Medina, M. W., et al, “Coordinately regulated alternative splicing of genes involved in cholesterol biosynthesis and uptake,” PLosONE 6: e19420 (2011)). This effect also extends to other cholesterogenic genes, including HMGSC1 and MVK (Id citing Medina, M. W., et al, “Coordinately regulated alternative splicing of genes involved in cholesterol biosynthesis and uptake,” PLosONE 6: e19420 (2011)). Because the effect is mediated via SREBP-2 and alternative transcripts occur for all cholesterol synthesis enzymes beyond HMGCR (Id., citing de la Grange, P., et al, “a new advance in alternative splicing databases from catalogue to detailed analysis of regulation of expression and function of human alternative splicing variants,” BMC Bioinformatics 8: 180 (2007)), this effect may involve the entire cholesterol synthesis pathway.
Post-translational Regulation
Because transcriptional down-regulation via the SREBP pathway is relatively slow, with mRNA of target genes decreasing only after several hours, rapid shutdown of cholesterol synthesis requires post-transcriptional control. Turnover of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) is accelerated by non-sterol and sterol products of the mevalonate pathways (Id., citing Roitelman, J. and Simoni, R D, “Distinct sterol and nonsterol signals for the regulated degradation of 3-hydroxy-3-methylglutaryl-CoA reductase,” J. Biol. Chem. 267: 25264-273 (1992)), with physiological sterol degradation signals, such as 24,25-dihydrolanosterol, and side chain oxysterols, such as 24,25-EC and 27-hydroxycholeseterol (generated from cholesterol itself (Id., citing Lange, Y. et al, “Effectors of rapid homeostatic responses of endoplasmic reticulum cholesterol and 3-hydroxy-3-methylglutaryl-CoA reductase,” J. Biol. Chem. 283: 1445-55 (2008); Nguyen, A D et al, “Hypoxyia stimulates degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase through accumulation of lanosterol and hypoxia-inducible factor-mediated induction of Insigs,” J. Biol. Chem. 282: 27436-446 (2007)). The regulated turnover is proteosomal, and requires the Insig proteins, which also act to suppress SREBP activation (Jo, Y and Debose-Boyd, R A, “Control of cholesterol synthesis through regulated ER-associated degradation of HMG CoA reductase,” Crit. Rev. Biochem. Mol. Bio. 445: 185-198 (2010); Burg, J S and Espenshade, P J, “Regulation of HMG-CoA reductase in mammals and yeast,” Prog. Lipid Res. 50: 403-410 (2011)).
Regulated ER-associated degradation also occurs for a later step in cholesterol synthesis, catalyzed by squalene monooxygenase (SM), albeit by a mechanism distinct from HMGCR. Squalene monooxygenase has been proposed as a second rate-limiting enzyme in cholesterol synthesis (Id., citing Gonzalez, R. et al, “Two major regulatory steps in cholesterol synthesis by human renal cancer cells,” Arch. Biochem. Biophys. 196: 574-80 (1979); Hidaka, Y, et al, “Regulation of squalene epoxidase in HepG2 cells,” J. Lipid Res. 31: 2087-94 (1990)). Cholesterol itself accelerates SM degradation, an example of end product inhibition (Id., citing Gill, S. et al, “Cholesterol-dependent degradation of squalene monooxygenase, a control point in cholesterol synthesis beyond HMG-CoA reductase,” Cell Metab. 13: 260-73 (2011)), and unlike HMGCR, SM turnover does not require the Insig proteins.
Feedback Regulation of Cholesterol Synthesis
Cholesterol accumulation lowers the activity of HMG CoA reductase and several other enzymes in the cholesterol biosynthetic pathway, thereby limiting the production of cholesterol.
HMG CoA reductase, the rate-limiting enzyme in cholesterol synthesis, and the target of statins, is subject to feedback control through multiple mechanisms that are mediated by sterol and nonsterol end-products of mevalonate metabolism such that essential nonsterol isoprenoids can be constantly supplied without risking the potentially toxic overproduction of cholesterol or one of its sterol precursors. (DeBose-Boyd, R. A. “Feedback Regulation of Cholesterol Synthesis: Sterol-accelerated ubiquitination and degradation of HMG CoA Reductase,” Cell Res. 18 (6): 609-21 (2008)). For example, treatment of cultured cells with the statin Compactin, a competitive inhibitor of HMG-CoA reductase, blocks production of mevalonate, thereby reducing levels of sterol and nonsterol isoprenoids that normally govern this feedback regulation. Id. Cells respond to the inhibition of HMG-CoA reductase with a compensatory increase in the reductase due to the combined effects of enhanced transcription of the reductase gene, efficient translation of mRNA, and extended half-life of reductase protein. Id. Complete reversal of this compensatory increase in reductase requires regulatory actions of both sterol and nonsterol end-products of mevalonate metabolism. Id.
Sterols inhibit the activity of sterol regulatory element-binding proteins (SREBPs) and the low density lipoprotein (LDL)-receptor (Id., citing Horton, J D, et al, “SREBPs: activators of the complete program of cholesterol and fatty acid synthesis in the liver,” J. Clin. Invest. 109: 1125-31 (2002)). A nonsterol mevalonate-derived product(s) control(s) the translational effects through a poorly understood mechanism that may be mediated by the complex 5′-untranslated region of the reductase mRNA (Id., citing Nakanishi, M. et al, “Multivalent control of 3-hydroxy-3-methylglutaryl coenzyme A reductase. Mevalonate-derived product inhibits translation of mRNA and accelerates degradation of enzyme,” J. Biol. Chem. 263: 8929-37 (1988)). Both sterol and nonsterol end-products of mevalonate metabolism combine to accelerate degradation of reductase protein through a mechanism mediated by the ubiquitin-proteosome pathway (Id., citing Roitelman, J. and Simoni, R D, “Distinct sterol and nonsterol signals for the regulated degradation of 3-hydroxy-3-methylglutaryl-CoA reductase,” J. Biol. Che. 267: 25264-273 (1992); McGee, T P et al, “Degradation of 3-hydroxy-3-methylglutaryl-CoA reductase in endoplasmic reticulum membranes is accelerated as a result of increased susceptibility to proteolysis,” J. Biol. Chem. 271: 25630-638 (1996); Ravid, T. et al, “The ubiquitin proteasome pathway mediates the regulated degradation of mammalian 3-hydroxy-3-methylglutaryl-Coenzyme A reductase,” J. Biol. Chem. 275: 35840-47 (2000)).
Inhibition of ER to Golgi transport of SREBPs results from sterol-induced binding of SCAP to ER retention proteins called insulin-induced gene 1 and 2 proteins (Insig-1 and Insig-2) (DeBose-Boyd, R. A. “Feedback Regulation of Cholesterol Synthesis: Sterol-accelerated ubiquitination and degradation of HMG CoA Reductase,” Cell Res. 18 (6): 609-21 (2008., citing Yang, T. et al, “Crucial step in cholesterol homeostasis: sterols promote binding of SCAP to INSIG-1, a membrane protein that facilitates retention of SREBPs in ER,” Cell 110: 489-500 (2002); Yabe, D. et al, “Insig-2, a second endoplasmic reticulum protein that binds SCAP and blocks export of sterol regulatory element-binding proteins,” Proc. Natl. Acad. Sci. USA 99: 12753-758 (2002)). Insig binding occludes a cytosolic binding site in SCAP recognized by COPII proteins, which incorporate cargo molecules into vesicles that deliver ER-derived proteins to the Golgi (Id., citing Sun L P et al, “From the Cover: Sterol-regulated transport of SREBPs from endoplasmic reticulum to Golgi: Insig renders sorting signal in Scap inaccessible to COPII proteins,” Proc. Natl Acad. Sci. USA 104: 6519-26 (2007)). SCAP-Insig binding is mediated by a segment of SCAP's membrane domain that includes transmembrane helices 2-6 (Id., citing Hua, X et al, “Sterol resistance in CHO cells traced to point mutation in SREBP cleavage-activating protein,” Cell 87: 415-26 (1996); Yang, T. et al, “Crucial step in cholesterol homeostasis: sterols promote binding of SCAP to INSIG-1, a membrane protein that facilitates retention of SREBPs in ER,” Cell 110: 489-500 (2002)), i.e., the sterol-sensing domain (Id., citing Kuwabara, P E, “The sterol-sensing domain: multiple families, a unique role,” Trends Genet. 18: 193-201 (2002)), since a similar stretch of transmembrane helices is found in at least four other polytopic proteins, including the Niemann Pick C1 protein (part of an intestinal cholesterol transporter complex), Patched, Dispatched and reductase) that have been postulated to interact with sterols. Point mutations within this region disrupt Insig binding, which relieves sterol-mediated retention of mutant SCAP-SREBP complexes in the ER (Id., citing Yang, T. et al, “Crucial step in cholesterol homeostasis: sterols promote binding of SCAP to INSIG-1, a membrane protein that facilitates retention of SREBPs in ER,” Cell 110: 489-500 (2002); Yabe, D., “Insig-2, a second endoplasmic reticulum protein that binds SCAP and blocks export of sterol regulatory element-binding proteins,” Proc. Natl. Acad. Sci. USA 99: 12753-758 (2002); Yabe, D. et al, “Three mutations in sterol-sensing domain of SCAP block interaction with insig and render SREBP cleavage insensitive to sterols,” Proc. Natl Acad. Sci. USA 99: 16672-77 (2002); Nohturfft, A. et al, “A substitution in a single codon of SREBP cleavage-activating protein causes sterol resistance in three mutant Chinese hamster ovary cell lines,” Proc. Natl Acad. Sci. USA 93: 13709-714 (1996); Nohturfft, A. et al, “Sterols regulate processing of carbohydrate chains of wild-type SREBP cleavage-activating protein (SCAP), but not sterol-resistant mutants Y298C o D443N,” Proc. Natl Acad. Sci. USA 95: 12848-853 (1998)).
The following observations suggest that Insigs may play a role in degradation of HMG CoA reductase. First, when Insigs are overexpressed by transfection in Chinese hamster ovary (CHO) cells, HMG CoA reductase cannot be degraded when the cells are treated with sterols (Id., citing Sever, N. et al, “Accelerated degradation of HMG CoA reductase mediated by binding of insig-1 to its sterol-sensing domain,” Mol. Cell 11: 25-33 (2003)). Co-expression of Insig-1 restores sterol-accelerated degradation of HMG CoA reductase, suggesting the saturation of endogenous Insigs by the overexpressed reductase. Id. Second, reduction of both Insig-1 and Insig-2 by RNA interference (RNAi) abolishes sterol-accelerated degradation of endogenous HMG CoA reductase (Id., citing Sever, N. et al, “Insig-dependent ubiquitination and degradation of mammalian 3-hydroxy-3-methylglutaryl CoA reductase stimulated by sterols and geranylgeraniol,” J. Biol. Chem. 278: 52479-90 (2003)). Third, mutant CHO cells lacking both Insigs are impervious to sterol-stimulated degradation of HMG CoA reductase as well as sterol-mediated inhibition of SREBP processing (Id., citing Lee, P. C. et al, “Isolation of sterol-resistant Chinese hamster ovary cells with genetic deficiencies in both Insig-1 and Insig-2,” J. Biol. Chem. 280: 25242-249 (2005)).
Degradation of HMG CoA reductase coincides with sterol-induced binding of its membrane domain to Insigs (Id., citing Sever, N. et al, “Accelerated degradation of HMG CoA reductase mediated by binding of insig-1 to its sterol-sensing domain,” Mol. Cell 11: 25-33 (2003)), an action that requires a tetrapeptide sequence (YIYF) located in the second transmembrane segment of HMG CoA reductase. A mutant form of HMG CoA reductase in which the YIYF sequence is mutated to alanine residues no longer binds to Insigs, and the enzyme is not subject to rapid degradation. The YIYF sequence is also present in the second transmembrane domain of SCAP, where it mediates sterol-dependent formation of SCAP-Insig complexes (Id., citing Yang, T. et al, “Crucial step in cholesterol homeostasis: sterols promote binding of SCAP to INSIG-1, a membrane protein that facilitates retention of SREBPs in ER,” Cell 110: 489-500 (2002); Yabe, D., “Insig-2, a second endoplasmic reticulum protein that binds SCAP and blocks export of sterol regulatory element-binding proteins,” Proc. Natl. Acad. Sci. USA 99: 12753-758 (2002)). Overexpressing the sterol-sensing domain of SCAP in cells blocks Insig-mediated, sterol-accelerated degradation of HMG CoA reductase; mutation of the YIYF sequence in the SCAP sterol-sensing domain ablates this inhibition, suggesting that SCAP and HMG CoA reductase bind to the same site on Insigs and that the two proteins compete for limiting amounts of Insigs when intracellular sterol levels rise. Id.
Glycoprotein 78 (Gp78), an E3 ubiquitin ligase, mediates ubiquitination of ApoB-100, Insig 1 and 2 proteins, and HMG-CoA reductase (Jiang, W., Song, B-L, “Ubiquitin Ligases in Cholesterol Metabolism,” Diabetes Metab. 38: 171-80 (2014)). High concentration of sterol (lanosterol) promote the NH2-terminal transmembrane domain of 3-hydroxy-3-methylglutaryl CoA reductase to interact with Insigs (Id., citing Sever, N. et al, “Accelerated degradation of HMG CoA reductase mediated by binding of insig-1 to its sterol-sensing domain,” Mol. Cell 11: 25-33 (2003); Song, B L, et al, “Insig-mediated degradation of HMG CoA reductase stimulated by lanosterol, an intermediate in the synthesis of cholesterol,” Cell Metab. 1: 179-89 (2005)), and sterol-dependent Insig binding results in recruitment of ubiquitin ligase.
Gp78 binds Insig-1 constitutively in the ER membrane.Id. When the cellular sterol level is high, the insig-1/gp78 complex binds the transmembrane domain of 3-hydroxy-3-methylglutaryl CoA reductase. Id. With the assistance of at least two proteins associated with gp78, p97/VCP and Aup1 (Id., citing Song, B L et al, “Gp8, a membrane-anchored ubiquitin ligase, associates with Insig-1 and couples sterol-regulated ubiquitination to degradation of HMG CoA reductase,” Mol. Cell 19: 829-40 (2005); Jo, Y et al, “ancient ubiquitous protein 1 mediates sterol-induced ubiquitination of 3-hydroxy-3-methylglutaryl CoA reductase in lipid droplet-associated endoplasmic reticulum membranes,” Mol. Biol. Cell 24: 169-83 (2013)), the ubiquitinated reductase is translocated to lipid droplet-associated ER membrane and dislocated from membrane into cytosol for proteosomal degradation (Id., citing Jo, Y et al, “ancient ubiquitous protein 1 mediates sterol-induced ubiquitination of 3-hydroxy-3-methylglutaryl CoA reductase in lipid droplet-associated endoplasmic reticulum membranes,” Mol. Biol. Cell 24: 169-83 (2013); Hartman I Z, et al, “Sterol-induced dislocation of 3-hydroxy-3-methylglutaryl coenzyme A reductase from endoplasmic reticulum membranes into the cytosol through a subcellular compartment resembling lipi droplets,” J. Biol. Chem. 285: 19288-98 (2010)). This post-ubiquitination process can be promoted by geranylgeraniol or its metabolically active geranyl-geranyl-pyrophosphate (Id., citing Sever, N. et al, “Insig-dependent ubiquitination and degradation of mammalian 3-hydroxy-3-methylglutaryl CoA reductase stimulated by sterols and geranylgeraniol,” J. Biol. Chem. 278: 52479-90 (2003)).
In short, the ubiquitination of Insig-1 is mediated by gp78 and regulated by sterols. Id. Insig-1 is modified by gp78 under low sterol conditions. Id. High sterol promotes SCAP to bind Insig and gp78 is competed off, thereby stabilizing Insig-1. Id.
Gp78-mediated ubiquitination and degradation of Insig-1 provides a mechanism for convergent feedback inhibition, whereby inhibition of SREBP processing requires convergence of newly synthesized Insig-1 and newly acquired sterols (DeBose-Boyd, R. A. “Feedback Regulation of Cholesterol Synthesis: Sterol-accelerated ubiquitination and degradation of HMG CoA Reductase,” Cell Res. 18 (6): 609-21 (2008); citing Gong, Y. et al, “Sterol-regulated ubiquitination and degradation of Insig-1 creates a convergent mechanism for feedback control of cholesterol synthesis and uptake,” Cell Metab. 3: 15-24 (2006)). In sterol-depleted cells, SCAP-SREBP complexes no longer bind Insig-1, which in turn becomes ubiquitinated and degraded. Id. These SCAP-SREBP complexes are free to exit the ER and translocate to the Golgi, where the SREBPs are processed to the nuclear form that stimulates transcription of target genes, including the Insig-1 gene. Id. Increased transcription of the Insig-1 gene leads to increased synthesis of Insig-1 protein, but the protein is ubiquitinated and degraded until sterols build up to levels sufficient to trigger SCAP binding. Id.
Insig-2 has been defined as a membrane-bound oxysterol binding protein with binding specificity that correlates with the ability of oxysterols to inhibit SREBP processing (Id., citing Sun, L P, et al, “Sterol-regulated transport of SREBPs from endoplasmic reticulum to Golgi: Insig renders sorting signal in Scap inaccessible to COPII proteins,” Proc. Natl Acad. Sci. USA 104: 6519-26 (2007); Radhakrishnan, A. et al, “From the Cover: Sterol-regulated transport of SREBPs from endoplasmic reticulum to Golgi: Oxysterols block transport by binding to Insig,” Proc. Natl Acad. Sci. USA 104: 6511-18 (2007)). Oxysterols, cholesterol derivatives that contain hydroxyl groups at various positions in the iso-octyl side chain (e.g., 24-hydroxycholesterol, 25-hydroxycholesterol, 27-hydroxycholesterol), are synthesized in many tissues by specific hydrolases; oxysterols play key roles in cholesterol export, and are intermediates in the synthesis of bile acids (Id., citing Russell, D W, “Oxsterol biosynthetic enzymes,” Biochim. Biophys. Acta—Molec. Cell Biol. Lipids 1529: 126-135 (2000)). Oxysterols, which are significantly more soluble than cholesterol in aqueous solution, can readily pass across the plasma membrane and enter cells, and are extremely potent in inhibiting cholesterol synthesis by stimulating binding of both HMG Co A reductase and SCAP to Insigs. Id. Thus, formation of the SCAP-Insig complex can be initiated by either binding of cholesterol to the membrane domain of SCAP or by binding of oxysterols to Insigs, both of which prevent incorporation of SCAP-SREBP into vesicles that bud from the ER en route to the Golgi. Id.
Insig-mediated regulation of HMG Co A reductase is controlled by three classes of sterols: oxysterols, cholesterol, and methylated sterols (e.g., lanosterol and 24, 25-dihydrolanosterol). Id. Oxysterols both accelerate degradation of HMG Co A reductase and block ER to Golgi transport of SCAP-SREBP through their direct binding to Insigs. Id. Cholesterol does not regulate HMG Co A reductase stability directly, but binds to SCAP and triggers Insig binding, thereby preventing escape of SCAP-SREBP from the ER.Id. Lanosterol selectively accelerates degradation of HMG Co A reductase without an effect on ER to Golgi transport of SCAP-SREBP. Id. However, the demethylation of lanosterol has been implicated as a rate-limiting step in the post-squalene portion of cholesterol synthesis (Id., citing Gaylor, J L, “Membrane bound enzymes of cholesterol synthesis from lanosterol,” Biochem. Biophys. Res. Communic., 292: 1139-46 (2002); Williams, M T, et al, “Investigation of the rate-determining microsomal reaction of cholesterol biosynthesis from lanosterol in Morris hepatomas and liver,” Cancer Res. 37: 1377-83 (1977)). The accumulation of lanosterol is avoided; its inability to block SREBP processing through SCAP assures that mRNAs encoding enzymes catalyzing reactions subsequent to lanosterol remain elevated, and lanosterol is metabolized to cholesterol.
It is a paradox that gp78 deficiency increases both the 3-hydroxy-3-methylglutaryl CoA reductase and Insig protein levels in mouse liver, because Insigs not only negatively regulate 3-hydroxy-3-methylglutaryl CoA reductase post-transcriptionally, but also inhibit SREBPs processing through binding to SCAP (Jiang, W. and Song, B-L, “Ubiquitin Ligases in Cholesterol Metabolism,” Diabetes Metab. 38: 171-80 (2014) citing Nohturfft, A. et al., “Topology of SREBP cleavage-activating protein, a polytopic membrane protein with a sterol sensing domain,” J. Biol. Chem. 273: 17243-250 (1998)). These two outcomes are contradictory regarding cholesterol biosynthesis. Studies from L-gp78+ mice have shown that the biosynthesis of cholesterol and fatty acids is decreased in gp78-deficient mouse liver (Id., citing Edwards, P A et al, “Purification and properties of rat liver 3-hydroxy-3-methylglutaryl coenzyme A reductase,” Biochim. Biophys. Acta 574: 123-35 (1979)). This has been interpreted to mean that the Insig-SCAP-SREBP axis dominates, even though 3-hydroxy-3-methylglutaryl CoA (HMG CoA) reductase is elevated. Id.
ApoB-100, an essential protein component of very low density lipoproteins (VLDL) and low density lipoproteins (LDL), which plays critical roles in plasma cholesterol transportation, is another substrate of g78. Id. Under normal conditions, ApoB-100 is one of the committed secretory proteins.Id. However, when the cellular lipid availability is limited (e.g., the new synthesized core lipids (triglyceride, cholesterol ester) or microsomal triglyceride transfer protein activity is decreased), the nascent ApoB-100 is subjected to ER-associated degradation mediated by gp78. Id. When gp78 is overexpressed, ubiquitination and degradation through the 26S proteosome of apoB-100 is decreased (Id., citing Ravid, T. et al, “The ubiquitin-proteasome pathway mediates the regulated degradation of mammalian 3-hydroxy-3-methylglutaryl-coenzyme A reductase,” J. Biol. Chem. 275: 35840-847 (2000)). When gp78 is knocked down, the secretion of apoB-100 and the assembly of VLDL are increased in HepG2 cells (Id., citing Hua, X., et al, “Sterol resistance in CHO cells traced to point mutation in SREBP cleavage-activating protein,” Cell 87: 415-426 (1996)). The retrotranslocation of ApoB-100 also requires p97/VCP, similar to HMG CoA reductase (Id, citing Nakanishi, M. et al, “multivalent control of 3-hydroxy-3-methylglutaryl coenzyme A reductase. Mevalonate-derived product inhibits translation of mRNA and accelerates degradation of enzyme,” J. Biol. Chem. 263: 8929-37 (1988); Hua, X., et al, “Sterol resistance in CHO cells traced to point mutation in SREBP cleavage-activating protein,” Cell 87: 415-426 (1996)).
TRC8
Human TRC8 is a multi-pass membrane protein located in the ER membrane that binds both Insig-1 and Insig-2. (Id., citing Inoue, S. et al, “Inhibition of degradation of 3-hydroxyl-3-methylglutaryl-coenzyme A reductase in vivo by cysteine protease inhibitors,” J. Biol. Chem. 266: 13311-17 (1991)). It contains a conserved sterol sensing domain and C-terminal RING domain with ubiquitin ligase activity (Id., citing Yabe, D. et al, “Insig-2, a second endoplasmic reticulum protein that binds SCAP and blocks export of sterol regulatory element-binding proteins,” Proc. Natl. Acad. Sci. USA 99: 12753-758 (2002); Sun L P et al, “From the Cover: Sterol-regulated transport of SREBPs from endoplasmic reticulum to Golgi: Insig renders sorting signal in Scap inaccessible to COPII proteins,” Proc. Natl Acad. Sci. USA 104: 6519-26 (2007)). RNAi studies in SV-589 cells showed that knockdown of TRC8 combined with gp78 can dramatically decrease the sterol-regulated ubiquitination as well as degradation of HMG CoA reductase, suggesting that both gp78 and TRC8 are involved in the sterol-accelerated ubiquitination of HMG CoA reductase in CHO-7 and SV-589 cells. (Id., citing Inoue, S. et al, “Inhibition of degradation of 3-hydroxyl-3-methylglutaryl-coenzyme A reductase in vivo by cysteine protease inhibitors,” J. Biol. Chem. 266: 13311-17 (1991)).
TEB4
Human TEB4 is a 910 amino acid ER membrane-resident ubiquitin ligase. In mammalian cells, cholesterol stimulates the degradation of squalene monooxygenase (SM), the enzyme that catalyzes the first oxygenation step in cholesterol synthesis by which squalene is converted to the squalene-2,3-epoxide (37) mediated by TEB4 (Id., citing Sever, N. et al, “Accelerated degradation of HMG CoA reductase mediated by binding of insig-1 to its sterol-sensing domain,” Mol. Cell 11: 25-33 (2003)). As one of the target genes of SREBP-2, both the transcription of SM and the stability of SM protein are regulated by sterols (Id., citing Sever, N. et al, Insig-dependent ubiquitination and degradation of mammalin 3-hydroxy-3-methylglutaryl-CoA reductase stimulated by sterols and geranylgeraniol,” J. Biol. Chem. 278: 52479-490 (2003)). SM protein level is negatively regulated by cholesterol in mammalian cells (Id., citing Lee, P C, et al, “Isolation of sterol-resistant Chinese hamster ovary cells with genetic deficiencies in both Insig-1 and Insig-2,” J. Biol. Chem. 280: 25242-249 (2005)). When cholesterol, but not 24, 25-dihydrolanosterol, or side chain oxysterols, such as 27-hydroxycholesterol, is/are present, SM is ubiquitinated by TEB4 (Id., citing Sever, N. et al, “Accelerated degradation of HMG CoA reductase mediated by binding of insig-1 to its sterol-sensing domain,” Mol. Cell 11: 25-33 (2003); Lee, P C, et al, “Isolation of sterol-resistant Chinese hamster ovary cells with genetic deficiencies in both Insig-1 and Insig-2,” J. Biol. Chem. 280: 25242-249 (2005)).
IDOL
The low density lipoprotein receptor (LDL-R) gene family consists of cell surface proteins involved in receptor-mediated endocytosis of specific ligands. Low density lipoprotein (LDL) is normally bound at the cell membrane and taken into the cell, ending up in lysosomes where the protein is degraded and the cholesterol is made available for repression of microsomal enzyme HMG CoA reductase. At the same time, a reciprocal stimulation of cholesterol ester synthesis takes place.
Inducible degrader of LDL-R (IDOL) moderates the degradation of LDL-R and requires the E2 enzyme UBE2D (Id., citing Schroepfer, G J, Jr., “Oxysterols: modulators of cholesterol metabolism and other processes,” Physiol. Rev. 80: 361-554 (2000); Bjorkhem, I., “Do oxysterols control choleseterol homeostasis,” J. Clin. Invest. 110: 725-30 (2002)).
Transcription of the LDL-R gene is regulated primarily by SREBP in a sterol responsive manner. (Id.) The LDL-R is also regulated at the posttranscriptional level by protoprotein convertase subtilisin/kexin type 9 (PCSK9)-mediated degradation of LDLR in the lysosome. (Id., citing Radhakrishnan, A. et al, “Direct binding of cholesterol to the purified membrane region of SCAP: Mechanism for a sterol-sensing domain,” Mol. Cell 15: 259-68 (2004)). PCSK9 is synthesized as an about 74 kD soluble zymogen in the endoplasmic reticulum (ER), where it undergoes autocatalytic processing to release a processing enzyme of about 60 kDa to secrete from cells. (Id.) PCSK9 binds the extracellular domain of LDLR, which leads to lysosomal degradation of LDLR. (Id.)
IDOL also is a post-transcriptional regulator of LDL-R (Id., citing Schroepfer, G J, Jr., “Oxysterols: modulators of cholesterol metabolism and other processes,” Physiol. Rev. 80: 361-554 (2000)). Activation of LXR can decrease the abundance of LDLR without changing its mRNA level and subsequently inhibited uptake of LDL in different cells (Id., citing Schroepfer, G J, Jr., “Oxysterols: modulators of cholesterol metabolism and other processes,” Physiol. Rev. 80: 361-554 (2000)). IDOL can increase plasma cholesterol level by ubiquitination and degradation of LDL-R dependent on its cytosolic domain. The decrease or ablation of IDOL can elevate the LDL-R protein level and promote LDL uptake. The expression of Idol in liver is relatively low, and it is not regulated by LXR, while the LXR-IDOL pathway seems to be more active in peripheral cells, e.g., macrophages, small intestine, adrenals.
Cholesterol Biosynthesis Pathway Inhibitors as Antitumor Agents
Statins, which were developed as lipid-lowering drugs to control hypercholesterolemia, competitively inhibit HMG-CoA reductase, and have been proposed as anticancer agents, because of their ability to trigger apoptosis in a variety of tumor cells in a manner that is sensitive and specific to the inhibition of HMG-CoA reductase (Thumher, M., et al., “Novel aspects of mevalonate pathway inhibitors as antitumor agents,” Clin. Cancer Res. 18: 3524-31 (2012) citing Wong, W W et al, “HMG-CoA reductase inhibitors and the malignant cell: the statin family of drugs as triggers of tumor-specific apoptosis,” Leukemia 16: 508-19 (2002)). This apoptotic response is in part due to the downstream depletion of geranylgeranyl pyrophosphate (GGPP), and thus due to inhibition of protein prenylation. Protein prenylation creates a lipidated hydrophobic domain and plays a role in membrane attachment or protein-protein interactions. Prenylation occurs on many members of the Ras and Rho family of small guanosine triphosphatases (GTPases). Three enzymes (farnesyltransferase (FTase), geranylgeranyltransferase (GGTase) I and GGTase II can catalyze protein prenylation.
While statin therapy blocks the intracellular synthesis of cholesterol, it also alters the cholesterol content of tumor cell membranes, interfering with key signaling pathways. (Cruz, P M R, et al, “The role of cholesterol metabolism and cholesterol transport in carcinogenesis: a review of scientific findings, relevant to future cancer therapeutics,” Frontiers in Pharmacol. 4(119): doi:10.3369/phar.2013.00119, citing Zhuang, L. et al, “Cholesterol targeting alters lipid raft composition and cell survival in prostate cancer cells and zenografts,” J. Clin. Invest. 115: 959-68 (2005)).
Statins have been shown to have immunomodulatory activity (Thumher, M., et al., “Novel aspects of mevalonate pathway inhibitors as antitumor agents,” Clin. Cancer Res. 18: 3524-31 (2012), citing Greenwood, J et al, Statin therapy and autoimmune disease: from protein prenylation to immunomodulation,” Nat. Rev. Immunol. 6: 358-70 (2006)), and to induce the depletion of prenyl pyrophosphates in human dendritic cells [Gruenbacher, G. et al., “CD56+ human blood dendritic cells effectively promote TH1-type gammadelta T cell responses,” Blood 114: 4422-31 (2009); Steinman, R M, Banchereau, J., “Taking dendritic cells into medicine,” Nature 449: 419-26 (2007)). Prenyl pyrophosphate deprivation translated into activation of caspase I, which cleaved the preforms of IL-1β and IL-18 and enabled the release of bioactive cytokines. The statin-treated dendritic cells (DCs) thus acquired the capability to potentially activate IL-2 primed natural killer (NK) cells (Id., citing Gruenbacher, G. et al., “IL-2 costimulation enables statin-mediated activation of human NK cells, preferentially through a mechanism involving CD56+ dendritic cells,” Cancer Res. 70: 9611-20 (2010)). NK cells, which recognize and attack tumor cells that lack MHC class I molecules (Id., citing Munz, C. et al, “Dendritic cell maturation by innate lymphocytes: coordinated stimulation of innate and adaptive immunity,” J. Exptl Med. 202: 203-7 (2005); Maniar, A. et al, “Human gammadelta T lymphocytes induce robust NK cell-mediated antitumor cytotoxicity through CD137 engagement,” Blood 116: 1726-33 (2010)) contribute to innate immune responses against neoplastic cells. The statin-induced response of IL-2-primed NK cells could be abolished completely when cell cultures were reconstituted with the isoprenoid pyrophosphate GGPP, which allows protein geranylgeranylation to occur despite statin-mediated inhibition of HMB-CoA reductase. Statins also acted directly on human carcinoma cells to induce apoptosis, and IFN-γ produced by NK cells cooperated with statins to enhance tumor cell death synergistically (Id., citing Gruenbacher, G. et al., “IL-2 costimulation enables statin-mediated activation of human NK cells, preferentially through a mechanism involving CD56+ dendritic cells,” Cancer Res. 70: 9611-20 (2010)).
Mutant p53, which is present in more than half of all human cancers, can significantly upregulate mevalonate pathway activity in cancer cells, which contributes to maintenance of the malignant phenotype. (Id., citing Freed-Pastor, W A, et al, “Mutant p53 disrupts mammary tissue architecture via the mevalonate pathway,” Cell 148: 244-58 (2012)). Simvastatin was shown to reduce 3-dimensional growth of cancer cells expressing a single mutant p53 allele, and was able to induce extensive cancer cell death and a significant reduction of their invasive phenotype. In isoprenoid add-back experiments, supplementation with GGPP was sufficient to restore the invasive phenotype in the presence of HMG-CoA reductase inhibition, showing that upregulation of protein geranylgeranylation is an important effect of mutant p53 (Id., citing Freed-Pastor, W A, et al, “Mutant p53 disrupts mammary tissue architecture via the mevalonate pathway,” Cell 148: 244-58 (2012)).
Bisphosphonates, drugs that prevent bone resorption, act downstream of HMG-CoA reductase to inhibit farnesyl pyrophosphate (FPP) synthase. Both bisphosphonates and statins eventually cause FPP and GGPP deprivation and thus failure to perform farnesylation and geranylgeranylation of small GTPases of the Ras superfamily. With regard to bisphosphonates, the inhibition of Ras signaling due to the disruption of membrane anchoring of these GTPases eventually stops osteoclast-mediated bone resorption (Id., citing Konstantinopoulos, P A, et al, “Post translational modifications and regulation of the RAS superfamily of GTPases as anticancer targets,” Nat. Rev. Drug Discov. 6: 541-55 (2007)).
Suppressors of the mevalonate pathway also include the diverse isoprenoids (Cruz, P M R, et al, “The role of cholesterol metabolism and cholesterol transport in carcinogenesis: a review of scientific findings, relevant to future cancer therapeutics,” Frontiers in Pharmacol. 4(119): doi:10.3369/phar.2013.00119, Id., citing Mo, H and Elson, C E, “Studies of the isopreoid-mediated inhibition of mevalonate synthesis applied to cancer chemotherapy and chemoprevention,” Exp. Biol. Med. (Maywood) 229: 567-85 (2004)), mevalonate-derived secondary metabolites of plants (Bach, T J, “Some new aspects of isoprenoid biosynthesis in plants—a review,” Lipids 30: 191-202 (1995)). The potencies of isoprenoids in suppressing hepatic HMG-CoA reductase activity was found to be strongly correlated to their potencies in tumor suppression (Id., citing Elson, C E and Quereshi, A A, “Coupling the choleseterol—and tumor-suppressive actions of palm oil to the impact of its minor constituents on 3-hydroxy-3-methylglutaryl coenzyme A reductase activity,” Prostaglandins Leukot. Essent. Fatty Acids 52: 205-207 (1995)). The tocotrienols, vitamin E molecules, and “mixed isoprenoids” with a farnesol side chain, down-regulate HMG-CoA reductase activity in tumors and consequently induce cell cycle arrest and apoptosis (Id., citing Mo H and Elfakhani, C E, “Mevalonate-suppressive tocotrienols for cancer chemoprevention and adjuvant therapy, in Tocotrienols: Vitamin E beyond tocopherols, eds. R R. Wilson et al (Boca Raton: CRC Press), 135-149 (2013)). The growth-suppressive effect of tocotrienols was attenuated by supplemental mevalonate (Id., citing Hussein, D and Mo, H, “d-δ-tocotrienol-mediated suppression of the proliferation of human PANC-1, M1A PaCa2 and BxPC-3 pancreatic carcinoma cells,” Pancreas 38: e124-e136 (2009)).
Activity of azole antifungal compounds, such as ketoconazole, to block the function of several cytochrome P450 enzymes involved in cholesterol biosynthesis (e.g., CYP51A1, which catalyzes demethylation of lanosterol) and CYP17A1 (which mediates a step in the synthesis of androgens) has been utilized clinically to treat hormone refractory prostate cancer, and recently has been surpassed by abiraterone, a CYP17A1 antagonist. Gorin, A. et al., “Regulation of cholesterol biosynthesis and cancer signaling,” Curr. Op. Pharmcol. 12(6) 710-16 (2012); citing (4). Itraconazole has shown activity against medulloblastoma, via its inhibitory effects on Smoothened in the hedgehog pathway. (Id., citing Kim, J. et al, “Itraconazole, a commonly used antifungal that inhibits Hedgehog pathway activity and cancer growth,” Cancer Cell. 17(4): 388-99 (2010)), and suppression of angiogenesis via its interference with lysosomal cholesterol trafficking (Id., citing Xu, J. et al, “Cholesterol trafficking is required for mTPOR activation in endothelial cells,” Proc. Natl Acad. Sci. USA 107(10): 4764-69 (2010)). The anti-angiogenic effect of itraconazole, a well-established CYP51/ERG11 antifungal antibiotic, is exerted via inhibition of endosomal cholesterol trafficking and suppression of mTOR signaling (Id.).
In tumor cells, increased signaling activity of growth factor or steroid hormone receptors via PI3K/AKT and MAPK/ERK1/2 (Gorin, A. et al., Regulation of cholesterol biosynthesis and cancer signaling, Curr. Opin. Pharmacol. 12(6): 710-16 (2012), citing Menendez, J A and Lupu, R., Fatty acid synthase and the lipogenic phenotype in cancer pathogenesis,” Nat. Rev. Cancer 7(10): 763-77 (2007)), HIF-1α, p53 (Id., citing Oliverase, G. et al, “Novel anti-fatty acid synthase compounds with anti-cancer activity in Her2+ breast cancer,” Ann. N.Y. Acad. Sci. 1210: 86-92 (2010)) and sonic hedgehog (SHH) (Id., citing Bhatia, B. et al, “Sonic hedgehog signaling and malignant transformation of the cerebellar granule neuron precursor cells,” Oncogene 30(4): 410-22 (2011)) pathways modulate and activate SREBP-1, the main regulatory component of lipogenesis. It has been reported that inhibiting mTORC1 using rapamycin has little effect on SREBP-1 nuclear localization and its abundance, but inhibiting its upstream factors, like EGFR, PI3K and Akt, significantly decreases SREBP-1 N-terminal levels and diminishes its abundance in the nucleus (Guo, D et al, “Targeting SREBP-1 driven lipid metabolism to treat cancer,” Curr. Pharm Des. 20(15): 2619-26 (2014) citing Guo, D. et al, “EGFR signaling through han Akt-SREBP-1-dependent, rapamycin-resistant pathway sensitizes glioblastomas to antilipogenic therapy,” Science Signaling 2: ra82 (2009)). mTOR kinase inhibitor Torin-1 (Id., citing Peterson, T R et al, “DEPTOR is an mTOR inhibitor frequently overexpressed in multiple myeloma cells and required for their survival,” Cell 137: 873-86 (2009)), which inhibits both mTORC1 and mTORC2 activity (Id., citing Sabatini, D M, “mTOR and cancer: insights into a complex relationship,” Nat. Rev. Cancer 6: 729-34 (2006)), significantly decreased SREBP-1 abundance in the nucleus compared to the inhibition of mTORC1 alone by rapamycin (Id., citing Peterson, T R, et al, “mTOR complex I regulates lipin 1 localization to control the SREBP pathway,” Cell 146: 408-20 (2011), Hagiwara, et al, “Hepatic mTORC2 activates glycolysis and lipogenesis through Akt, glucokinase and SREBP1c,” Cell Metab. 15: 725-38 (2012)).
Overexpression of lipogenic enzymes has been observed in a number of carcinomas (Gorin, A. et al., Regulation of cholesterol biosynthesis and cancer signaling, Curr. Opin. Pharmacol. 12(6): 710-16 (2012), citing Nagahashi, M. et al, “Sphingosine-1-phosphate produced by sphingosine kinase 1 promotes breast cancer progression by stimulating angiogenesis and lymphangiogenesis,” Cancer Res. 72(3): 726-35 (2012)) and has been described to correlate with disease severity, increased risk of recurrence and a lower chance of survival (Id., citing Uddin, S. et al, “High prevalence of fatty acid synthase expression in colorectal cancers in Middle Eastern patients and its potential role as a therapeutic target,” Am. J. Gastroenterol. 104(7): 1790-1801 (2009; Mashima, T. et al, “De novo fatty-acid synthesis and related pathways as molecular targets for cancer therapy,” Br. J. Cancer 100 (9): 1369-72 (2009)).
Accelerated synthesis of lipids and sterols also is an essential mechanistic component of malignant transformation. Oxidized LDL receptor 1 (OLR1) is required for Src kinase transformation of immortalized MCF10A mammary epithelial cells (Id., citing Hirsch, H A et al, “A transcriptional signature and common gene networks link cancer with lipid metabolism and diverse human diseases,” Cancer Cell. 17(4): 348-61 (2010)). OLR1 is significantly induced during transformation, and depletion of OLR1 by siRNA blocks morphological transformation and inhibits cell migration and invasion, and results in reduction of tumor growth in vivo (Id.). Conversely, overexpression of ORL1 protein in MCF10A and HCC 1143 mammary epithelial cells leads to significant upregulating of BCL2, a negative regulator of apoptosis (Id., citing Khaidakov, M., et al., “Oxidized LDL receptor 1 (OLR1) as a possible link between obesity, dyslipidemia and cancer,” PLoS One 6(5): e20277 (2011)).
EBP in complex with dihydrocholesterol-7 reductase (DHCR7) catalyzes isomerization of the double-bond between C7 and C8 in the second cholesterol ring. (Gabitova, L. et al., “Molecular Pathways: Sterols and receptor signaling in Cancer,” Clin. Cancer Res. 19(23): 6344-50 (2013)). This complex mediates the activity of cholesterol epoxide hydrolase (Id., citing de Medina, P. et al, “Identification and pharmacological characterization of cholesterol-5,6-epoxide hydrolase as a target for tamoxifen and AEBS ligands,” Proc. Natl. Acad. Sci. USA 107: 13520-5 (2010)).
There are several known inhibitors of EBP, and some have been described as anti-cancer agents. For example, a sterol conjugate of a naturally occurring steroidal alkaloid, 5alpha-hydroxy-6beta-[2-(1H-imidazol-4-yl)ethylamino]cholestan-3beta-ol (dendrogenin A) which is produced in normal, but not in cancer cells, and 5,6 alpha-epoxy-cholesterol and histamine (Id., citing de Medina, P. et al, “Dendrogenin A arises from cholesterol and histamine metabolism and shows cell differentiation and anti-tumour properties,” Nature Communic. 4: 1840 (2013); de Medina, P. et al, “Synthesis of new alkylaminooxysterols with potent cell differentiating activities: identification of leads for the treatment of cancer and neurodegenerative diseases,” J. Med. Chem. 52: 7765-77 (2009)), has been shown to suppress cancer cell growth and to induce differentiation in vitro in various tumor cell lines of different types of cancers (Id., citing de Medina, P. et al, “Synthesis of new alkylaminooxysterols with potent cell differentiating activities: identification of leads for the treatment of cancer and neurodegenerative diseases,” J. Med. Chem. 52: 7765-77 (2009)). It also inhibited tumor growth in melanoma xenograft studies in vivo and prolonged animal survival. (Id., citing de Medina, P. et al, “Dendrogenin A arises from cholesterol and histamine metabolism and shows cell differentiation and anti-tumour properties,” Nature Communic. 4: 1840 (2013);).
SR31747A (cis-N-cyclohexyl-N-ethyl-3-(3-chloro-4-cyclohexyl-phenyl)propen-2-ylamine hydrochloride), a selective peripheral sigma binding site ligand whose biological activities include immunoregulation and inhibition of cell proliferation, binds to SR31747A-binding protein 1 (SR-BP) and EBP with nanomolar affinity. Berthois, Y. et all., “SR31747A is a sigma receptor ligand exhibiting antitumoural activity both in vitro and in vivo,” Br. J. Cancer 88: 438-46 (2003). The effect of SR31747A on proliferative activity was evaluated in vitro on the following breast and prostate cancer cell lines: breast (hormone responsive MCF-7 cells from a breast adenocarcinoma pleural effusion; MCF-7AZ; Hormone independent MCF-7/LCC1 cells derived from MCF-7 cell lines; MCF-7LY2, resistant to the growth-inhibitory effects of the antiestrogen LY117018; Hormone unresponsive MDA-MB-321 and BT20 established from a metastatic human breast cancer tumor); and prostate (Hormone responsive prostate cancer cell line LNCaP; hormone-unresponsive PC3 cell line established from bone marrow metastasis; hormone-unresponsive DU145 established from brain metastasis). Id. SR31747A induced concentration-dependent inhibition of cell proliferation, regardless of whether the cells were hormone responsive or unresponsive. Id. The antiproliferative effect of SR31747A was partially reduced by adding cholesterol (Id.; Labit-Le Bouteiller, C. et al., “Antiproliferative effects of SR31747A in animal cell lines are mediated by inhibition of cholesterol biosynthesis at the sterol isomerase step,” Eur. J. Biochem. 256: 342-49 (1998)), thus demonstrating the involvement of EBP. Sensitivity to SR31747A did not correlate with cellular levels of EBP. Berthois, Y. et all., “SR31747A is a sigma receptor ligand exhibiting antitumoural activity both in vitro and in vivo,” Br. J. Cancer 88: 438-46 (2003). SR31747A also inhibited proliferation in vivo in the mouse xenograft model. Id. Murine EBP cDNA overexpression in CHO cells increased resistance of these cells to SR31747A-induced inhibition of proliferation. Labit-Le Bouteiller, C. et al., “Antiprolifertive effects of SR31747A in animal cell lines are mediated by inhibition of cholesterol biosynthesis at the sterol isomerase step,” Eur. J. Biochem. 256: 342-49 (1998)),
Tamoxifen, inhibited SR31747 binding in a competitive manner and induced the accumulation of Δ8-sterols, while Emopamil, a high affinity ligand of human sterol isomerase, and verapamil, another calcium channel-blocking agent, are inefficient in inhibiting SR31747 binding to its mammalian target, suggesting that their binding sites do not overlap. Paul, R. et al., “Both the immunosuppressant SR31747 and the antiestrogen tamoxifen bind to an emopamil-insensative site of Mammalian A8-47 sterol isomerase,” J. Pharmacol. Exptl Thera. 285(3): 1296-1302 (1998)). Some drugs, e.g., cis-flupentixol, trifluoroperazine, 7-=ketocholestanol and tamoxifen, inhibit SR31747 binding only with mammalian EBP enzymes, whereas other drugs, e.g., haloperidol and fenpropimorph, are more effective with the yeast enzyme than with the mammalian ones. Id.
While some cancer cell lines are highly sensitive to small molecule EBP inhibition, other cancer cell lines, as well as normal cell lines, do not respond to EBP inhibition, even when up to 10,000-fold higher concentrations of the EBP inhibitors are used. A determination of which cancer will respond to which inhibitor therefore has required an empirical hit or miss, impractical and expensive, approach.
The described invention establishes that EBP inhibition is only toxic to cancer cells that paradoxically respond to small molecule EBP inhibitors via downregulation of endogenous cholesterol biosynthesis, and provides a method for identifying such EBP inhibitors and for cancer cells that are sensitive to treatment with such inhibitors.